There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

• MeSH
• Cell Division MeSH
• Cartilage MeSH
• Bone and Bones MeSH
• Osteogenesis * MeSH
• Mammals MeSH
• Caudata * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Lightless caves can harbour a wide range of living organisms. Cave animals have evolved a set of morphological, physiological, and behavioural adaptations known as troglomorphisms, enabling their survival in the perpetual darkness, narrow temperature and humidity ranges, and nutrient scarcity of the subterranean environment. In this study, we focused on adaptations of skull shape and sensory systems in the blind cave salamander, Proteus anguinus, also known as olm or simply proteus-the largest cave tetrapod and the only European amphibian living exclusively in subterranean environments. This extraordinary amphibian compensates for the loss of sight by enhanced non-visual sensory systems including mechanoreceptors, electroreceptors, and chemoreceptors. We compared developmental stages of P. anguinus with Ambystoma mexicanum, also known as axolotl, to make an exemplary comparison between cave- and surface-dwelling paedomorphic salamanders. FINDINGS: We used contrast-enhanced X-ray computed microtomography for the 3D segmentation of the soft tissues in the head of P. anguinus and A. mexicanum. Sensory organs were visualized to elucidate how the animal is adapted to living in complete darkness. X-ray microCT datasets were provided along with 3D models for larval, juvenile, and adult specimens, showing the cartilage of the chondrocranium and the position, shape, and size of the brain, eyes, and olfactory epithelium. CONCLUSIONS: P. anguinus still keeps some of its secrets. Our high-resolution X-ray microCT scans together with 3D models of the anatomical structures in the head may help to elucidate the nature and origin of the mechanisms behind its adaptations to the subterranean environment, which led to a series of troglomorphisms.

• Keywords
• Ambystoma mexicanum, Proteus anguinus, X-ray microCT, axolotl, cave animal, microtomography, olm, salamander, subterranean adaptations,
• MeSH
• Proteidae * MeSH
• X-Rays MeSH
• Darkness MeSH
• Caudata MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

3D imaging approaches based on X-ray microcomputed tomography (microCT) have become increasingly accessible with advancements in methods, instruments and expertise. The synergy of material and life sciences has impacted biomedical research by proposing new tools for investigation. However, data sharing remains challenging as microCT files are usually in the range of gigabytes and require specific and expensive software for rendering and interpretation. Here, we provide an advanced method for visualisation and interpretation of microCT data with small file formats, readable on all operating systems, using freely available Portable Document Format (PDF) software. Our method is based on the conversion of volumetric data into interactive 3D PDF, allowing rotation, movement, magnification and setting modifications of objects, thus providing an intuitive approach to analyse structures in a 3D context. We describe the complete pipeline from data acquisition, data processing and compression, to 3D PDF formatting on an example of craniofacial anatomical morphology in the mouse embryo. Our procedure is widely applicable in biological research and can be used as a framework to analyse volumetric data from any research field relying on 3D rendering and CT-biomedical imaging.

• MeSH
• Models, Anatomic MeSH
• Electronic Data Processing MeSH
• Data Compression statistics & numerical data   MeSH
• Skull anatomy & histology   embryology   MeSH
• Mice MeSH
• Facial Bones anatomy & histology   embryology   MeSH
• X-Ray Microtomography statistics & numerical data   MeSH
• Radiographic Image Interpretation, Computer-Assisted MeSH
• Information Dissemination methods   MeSH
• Software * MeSH
• Imaging, Three-Dimensional statistics & numerical data   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH